This proposal brings together investigators from Mayo Clinic, Rutgers University, and Walter Reed Army Institute of Research with expertise in computational and medicinal chemistry, structural biology, biochemistry, molecular biology and immunology to develop broad-spectrum therapy against ribosome inactivating proteins. Our primary goal is to develop second-generation small-molecule inhibitors of Shiga-like toxin 2 (Stx2) and ricin. Both toxins are category B agents that can cause significant morbidity and mortality. Currently, there is no FDA-approved vaccine or therapeutics that can protect against either toxin. Traditional approaches to ricin/Shiga toxin inhibitors use either a brute-force screening method to identify leads or a structure-based method to identify analogs of substrate adenine. The toxin-transport-system-targeting inhibitors with IC5O values of 25-50 ?M identified from the screening method suffer from the challenge in optimizing the leads into nanomolar inhibitors because of the lack of the structural information on the transport system. All adenine-like inhibitors reported to date are either small molecules with submillimolar potencies or oligonucleotides that can neutralize the extracellular toxin to prevent further intoxication but cannot effectively cross the plasma membrane to rescue intoxicated cells. This application innovatively uses the molecular "clip" approach that identifies small molecules able to lock TyrSo of ricin (or its equivalent Tyryy of Stx2) into the conformation that blocks the function of the protein. Such inhibitors do not have to be adenine-like for direct competition with RNA for binding to the toxin, thereby avoiding the permeability problem of the known ricin/Shiga inhibitors. This novel approach has already culminated in small molecules that inhibited both ricin and Stx2 at a drug concentration of 10 nM and showed ~io% cell protection against ricin and 20% cell protection against Stx2 at a drug concentration of 300 nM. In this context, we propose to optimize our inhibitor leads to improve their affinity for the active site of Stx2/ricin and their cell permeability, measure the potency of the inhibitors in antagonizing Stx2/ricin in cell free and cell based assays and examine the inhibitors in combination with a ricin vaccine under development for synergistic efficacy to prevent or alleviate ricin intoxication. The significant outcomes of this project are (i) small molecules that inhibit both Shiga and ricin at nanomolar drug concentrations in vivo, enabling a therapy effective against multiple toxins;(2) knowledge useful for identification and optimization of other small-molecule inhibitors of toxins that target ribosomes;(3) a proof-of- concept for use of small-molecule inhibitors as adjuncts to immunization to explore the synergy of small-molecule and vaccine therapies. Ultimately the work proposed here will result in the development several highly potent and specific inhibitors of toxin-mediated injury that are potential drug candidates. RELEVANCE (See instructions): The research is most relevant to public health and biodefense.